1. Field of the Invention
This invention relates to a measuring method and apparatus using attenuation in total internal reflection such as a surface plasmon sensor for analyzing a sample on the basis of generation of surface plasmon.
2. Description of the Related Art
In metal, free electrons vibrate in a group to generate compression waves called plasma waves. The compression waves generated in a metal surface are quantized into surface plasmon.
There have been proposed various surface plasmon sensors for quantitatively analyzing a material in a sample utilizing a phenomenon that such surface plasmon is excited by light waves. Among those, one employing a system called “Kretschmann configuration” is best known. See, for instance, Japanese Unexamined Patent Publication No. 6(1994)-167443.
The plasmon resonance sensor using the Kretschmann configuration basically comprises a dielectric block shaped, for instance, like a prism, a metal film which is formed on one face of the dielectric block and is brought into contact with a sample, a light source emitting a light beam, an optical system which causes the light beam to enter the dielectric block to impinge upon the interface of the dielectric block and the metal film at various angles of incidence so that total internal reflection conditions are satisfied at the interface, and a photodetector means which detects the intensity of the light beam reflected in total internal reflection at the interface and detects a state of surface plasmon resonance, i.e., a state of attenuation in total internal reflection.
In order to obtain various angles of incidence of the light beam to the interface, a relatively thin incident light beam may be caused to impinge upon the interface while deflecting the incident light beam or a relatively thick incident light beam may be caused to impinge upon the interface in the form of convergent light or divergent light so that components of the incident light beam impinge upon the interface at various angles. In the former case, the light beam which is reflected from the interface at an angle which varies as the incident light beam is deflected may be detected by a photodetector which is moved in synchronization with deflection of the incident light beam or by an area sensor extending in the direction in which reflected light beam is moved as a result of deflection. In the latter case, an area sensor which extends in directions so that all the components of light reflected from the interface at various angles can be detected by the area sensor may be used.
In such a plasmon resonance sensor, when a light beam impinges upon the interface at a particular angle of incidence θsp not smaller than the angle of, total internal reflection, evanescent waves having an electric field distribution in the sample in contact with the metal film are generated and surface plasmon is excited in the interface between the metal film and the sample. When the wave number vector of the evanescent waves is equal to the wave number of the surface plasmon and wave number matching is established, the evanescent waves and the surface plasmon resonate and light energy is transferred to the surface plasmon, whereby the intensity of light reflected in total internal reflection at the interface of the dielectric block and the metal film sharply drops. The sharp intensity drop is generally detected as a dark line by the photodetector. The aforesaid resonance occurs only when the incident light beam is p-polarized. Accordingly, it is necessary to set the light beam to impinge upon the interface in the form of p-polarized light.
When the wave number of the surface plasmon can be known from the angle of incidence θsp at which the phenomenon of attenuation in total internal reflection (ATR) takes place, the dielectric constant of the sample can be obtained. That is,
            K      sp        ⁡          (      ω      )        =            ω      c        ⁢                                                      ɛ              m                        ⁡                          (              ω              )                                ⁢                      ɛ            s                                                              ɛ              m                        ⁡                          (              ω              )                                +                      ɛ            s                              wherein Ksp represents the wave number of the surface plasmon, ω represents the angular frequency of the surface plasmon, c represents the speed of light in a vacuum, and ∈m and ∈s respectively represent the dielectric constants of the metal and the sample.
A property related to the dielectric constant ∈s (refractive index) of the sample can be detected by detecting the angle of incidence θsp at which the intensity of light reflected in total internal reflection from the interface of the prism and the metal film sharply drops (this angel θsp will be referred to as “the attenuation angle θsp”, hereinbelow).
In such a surface plasmon sensor, it has been proposed, in order to measure the attenuation angle θsp accurately with a wide dynamic range, to use a photodetector in the form of an array of a plurality of photodetector elements arranged in a predetermined direction so that light beam components reflected at different angles at the interface impinge upon different photodetector elements as disclosed in Japanese Unexamined Patent Publication No. 11 (1999)-326194.
In this case, the output signals output from the photodetector elements are generally differentiated in the direction in which the photodetector elements are arranged, and the refractive-index-related property of the material to be measured is generally obtained on the basis of the differentials.
As a similar apparatus utilizing the phenomenon of attenuation in total internal reflection (ATR), there has been known a leaky mode sensor described in, for instance, “Spectral Research” Vol.47, No.1 (1998), pp21 to 23 & pp26 and 27. The leaky mode sensor basically comprises a dielectric block shaped, for instance, like a prism, a clad layer which is formed on one face of the dielectric block, an optical waveguide layer which is formed on the clad layer and is brought into contact with a sample liquid, a light source emitting a light beam, an optical system which causes the light beam to enter the dielectric block to impinge upon the interface of the dielectric block and the metal film at various angles of incidence so that total internal reflection conditions are satisfied at the interface, and a photodetector means which detects the intensity of the light beam reflected in total internal reflection at the interface and detects a state of waveguide mode excitation, i.e., a state of attenuation in total internal reflection (ATR).
In the leaky mode sensor with this arrangement, when the light beam is caused to impinge upon the clad layer through the dielectric block at an angle not smaller than an angle of total internal reflection, only light having a particular wave number and impinging upon the optical waveguide layer at a particular angle of incidence comes to propagate through the optical waveguide layer in a waveguide mode after passing through the clad layer. When the waveguide mode is thus excited, almost all the incident light is taken in the optical waveguide layer and accordingly, the intensity of light reflected in total internal reflection at the interface of the dielectric block and the clad layer sharply drops. That is, attenuation in total internal reflection occurs. Since the wave number of light to be propagated through the optical waveguide layer in a waveguide mode depends upon the refractive index of the sample on the optical waveguide layer, the refractive index and/or the properties of the sample related to the refractive index can be detected on the basis of the angle of incidence at which the attenuation in total internal reflection occurs.
Also in such a leaky mode sensor, a photodetector in the form of an array of a plurality of photodetector elements can be used to detect the position of the dark line generated due to the attenuation in total internal reflection, and at the same time differentiation of the output signals output from the photodetector elements is often applied.
The surface plasmon sensor and the leaky mode sensor are sometimes used in random screening for finding a specific material combined with a predetermined sensing material in the field of pharmacy. In this case, a sensing material is fixed on the film layer (the metal film in the case of the surface plasmon sensor and the clad layer and the optical waveguide layer in the case of the leaky mode sensor), and a sample liquid containing a material to be analyzed is spotted on the sensing material. Then the attenuation angle θsp is repeatedly measured each time a predetermined time lapses.
When the sample material (the material to be analyzed in the sample liquid) is combined with the sensing material, the refractive index of the sensing material changes with time due to combination with the sample material. Accordingly, by measuring the attenuation angle θsp, at which attenuation in total internal reflection takes place, for every predetermined time, thereby detecting whether the attenuation angle θsp changes (to know the state of combination of the sample material with the sensing material), it is possible to know whether the sample material is a specific material to be combined with the sensing material. As combinations of such a specific material and a sensing material, there have been known combinations of an antigen and an antibody and of an antibody and another antibody. For example, rabbit antihuman IgG antibody is fixed on the surface of the film layer as the sensing material with human IgG antibody employed as the specific material.
In order to detect the state of combination of the sample material with the sensing material, the total reflection attenuation angle θsp (the angle of incidence θsp at which attenuation in total internal reflection takes place) itself need not necessarily be detected. For example, the amount of change in the total reflection attenuation angle θsp after the sample liquid is spotted onto the sensing material is measured and the state of combination of the sample material with the sensing material may be measured on the basis of the amount of change of the total reflection attenuation angle θsp. When a photodetector in the form of an array of a plurality of photodetector elements and differentiation of the output signals output from the photodetector elements are employed, the state of combination of the sample material with the sensing material can be measured on the basis of the amount of change of the differentiation of the output signals. (See our Japanese Patent Application 2000-398309.)
In the practical apparatuses utilizing the phenomenon of attenuation in total internal reflection such as a surface plasmon sensor or a leaky mode sensor, the amount of change of the total reflection attenuation angle θsp is measured by spotting a sample liquid comprising solvent and a sample material onto a film layer formed on the bottom of a measuring chip in the form of a cup or dish.
When a sample liquid is spotted on a measuring chip and a sample material in the sample liquid is combined with the sensing material, the refractive index of the sensing material changes and the attenuation angle θsp changes. However, strictly speaking, the change of the attenuation angle θsp does not solely depend upon the state of combination of the sample material with the sensing material but also depends upon action between the sensing material and the solvent of the sample liquid and difference in sensitivity among measuring apparatuses.
That is, even when a false sample liquid which solely consists of solvent and includes no sample material is supplied to a sensing material, the attenuation angle θsp slightly changes as shown by the solid line in FIG. 4. The change of the attenuation angle θsp produces a measuring error. In order to avoid production of this measuring error, we, this applicant has proposed, in our Japanese Patent Application 2001-049681, a method of judging whether a sensing material is combined with a sample material, in which a corrected attenuation angle change is obtained by subtracting the change of the attenuation angle θsp in a reference chip supplied with a false sample liquid from the change of the attenuation angle θsp in a measuring chip supplied with a real sample liquid and whether the sensing material is combined with a sample material in the sample liquid is judged on the basis of the corrected attenuation angle change. In accordance with the method, since the corrected attenuation angle change becomes substantially 0 when the sensing material is not combined with the sample material, whether the sensing material is combined with a sample material in the sample liquid can be easily judged.
However, this method is disadvantageous in that since the corrected attenuation angle change does not become completely 0 even if the sensing material is not combined with the sample material, this method is not effective when the sample material is small in molecular weight though effective when the sample material is large in molecular weight.
The attenuation angle change in a measuring chip supplied with a false sample liquid as measured by these inventors is as shown by the solid line in FIG. 4, whereas the attenuation angle change in a reference chip supplied with the same false sample liquid as measured by these inventors is as shown by the broken line in FIG. 4. The corrected attenuation angle change in this case is as shown by the chained line in FIG. 4. Since the sensing material is combined with the sample material in neither cases, the corrected attenuation angle should be 0. However the attenuation angle change after one hour shown by the chained line shows a value of 900 in terms of molecular weight. That is, the corrected attenuation angle, which should be 0, is too large, which deteriorates the measuring accuracy.
A reason why there is produced a difference between the attenuation angle change in a reference chip and that in a measuring chip supplied with the same false sample liquid is, for instance, the difference in thickness of the metal films between the measuring chip and the reference chip. Another reason may be a difference in sensitivity between the photodetector means of the measuring unit and that of the reference unit.
Further, this applicant has disclosed a measuring apparatus utilizing the phenomenon of attenuation in total internal reflection in which a plurality of measuring chips are placed on, for instance, a turn table and are measured in sequence, thereby shortening the time required to measure a lot of samples. (Japanese Unexamined Patent Publication No. 2001-330560)
Further, this applicant has disclosed a measuring apparatus utilizing the phenomenon of attenuation in total internal reflection in which measuring chips each having a plurality of sample liquid holding portions are used so that measurement on a plurality of samples can be done at one time without moving the measuring chips. (Japanese Patent Application 2001-397411)